Fig 2: The V-ATPase acts as a scaffold for SYK-mediated cGAS priming at the early endosomes. (A) Interaction of cGAS with components of the V-ATPase. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 20 h and then lysed for coimmunoprecipitation with HA antibody, followed by immunoblot analysis with the indicated antibodies. (B) Effects of overexpression of individual component of V-ATPase on association between human SYK and cGAS. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 20 h, and then lysed for coimmunoprecipitation with Flag antibody, followed by immunoblot analysis with the indicated antibodies. (C) Effects of V-ATPase inhibitor on human SYK-ATP6VA1 and SYK-cGAS association. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 12 h before cells were left untreated or treated with EN6 (30 µM) for 10 h, and then cells were lysed for coimmunoprecipitation with Flag antibody, followed by immunoblot analysis with the indicated antibodies. (D) Effects of V-ATPase inhibitors on cGAS activity. Raw264.7 cells were pretreated with DMSO, EN6 (30 µM), or Baf-A1 (20 nM) for half an hour, and then mock-transfected or transfected with DNA-Cy5 (1 µg/mL) for 4 h, followed by cGAMP activity assay. The DNA transfection efficiency in each group is displayed (Right). (E) Effects of V-ATPase inhibitors on endocytosed foreign DNA-induced transcription of the Ifnb1 gene. Raw264.7 cells (1 × 106) were pretreated with DMSO, EN6 (30 µM), or Baf-A1 (20 nM) for half an hour, and then treated with HSV-1 (MOI = 1) for 6 h, transfected with HT-DNA (1 µg/mL) for 4 h, or treated with A/Q (10 µM each) for 6 h, followed by qPCR analysis of the Ifnb1 gene. (F) Effects of V-ATPase inhibitors on viral infection-induced phosphorylation of Tbk1 and Irf3. Raw264.7 cells (1 × 106) were pretreated with DMSO, EN6 (30 µM), or Baf-A1 (20 nM) for half an hour, and then left uninfected or infected with HSV-1 (MOI = 2) for the indicated times, followed by immunoblot analysis with the indicated antibodies. (G and H) Effects of transient knockdown of Atp6v1a and Atp6v1d on HSV-1– or IFN-?–induced transcription of downstream genes. Raw264.7 cells (1 × 106) were transiently transfected with the indicated CRISPR-Cas9 vectors for 60 h, and then infected with HSV-1 (MOI = 1) for the indicated times (G) or treated with IFN-? (100 ng/mL) for 4 h (H), followed by qPCR analysis of the indicated genes. ns, nonsignificant; **P < 0.01 (unpaired t test). The data shown are the mean ± SD (n = 3). All experiments were performed at least two times.